Building eResearch Services, Capabilities and Capacity

The Queensland University of Technology (QUT) is known for several flagship eResearch centres. It also has a number of mature, centralised research support services that address a several of areas of eResearch. The university has openly stated its aspiration to be an institution with a strongly embedded eResearch capability and to this end it has expressed the desire to establish a university-wide eResearch support service. However, articulating this desire is much easier than realising it. During 2008 QUT undertook a major review into eResearch that made recommendations on the development of university-wide eResearch support service and the building of eResearch capabilities and capacity throughout the university. The results of this review were reported last year at this conference. In 2009, QUT is progressing a second, follow-on project – Building eResearch Support Capability and Capacity. It has been designed to build upon existing strengths in HPC, repositories, data management, and the delivery of integrated skills for eresearch. The purpose of this presentation is to give an update on QUT’s journey, one year on from its first major report into eResearch. It will outline how the university is approaching this challenge, the current work being carried out and the strategies being employed. We will also discuss the lessons learned.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Venture creation and resource processes : using Bricolage sustainability ventures

Principal Topic: Resource decisions are critical to the venture creation process, which has important subsequent impacts on venture creation and performance (Boeker, 1989). Most entrepreneurs however, suffer substantial resource constraints in venture creation and during venture growth (Shepherd et al., 2000). Little is known about how high potential, sustainability ventures (the ventures of interest in this research), despite resource constraints, achieve continued venture persistence and venture success. One promising theory that explicitly links to resource constraints is a concept developed by Levi Strauss (1967) termed bricolage. Bricolage aligns with notions of resourcefulness: using what's on hand, through making do, and recombining resources for new or novel purposes (Baker & Nelson 2005). To the best of our knowledge, previous studies have not systematically investigated internal and external constraints, their combinations, and subsequent bricolage patterns. The majority of bricolage literature focuses on external environmental constraints (e.g. Wieck 1989; Baker & Nelson 2005), thereby paying less attention to in evaluating internal constraints (e.g. skills and capabilities) or constraint combinations. In this paper we focus on ventures that typically face resource-poor environments. High potential, nascent and young sustainability ventures are often created and developed with resource constraints and in some cases, have greater resource requirements owing to higher levels of technical sophistication of their products (Rothaermel & Deeds 2006). These ventures usually have high aspirations and potential for growth who ''seeks to meet the needs and aspirations without compromising the ability to meet those of the future'' (Brundtland Commission 1983). High potential ventures are increasingly attributed with a central role in the development of innovation, and employment in developed economies (Acs 2008). Further, increasing awareness of environmental and sustainability issues has fostered demand for business processes that reduce detrimental environmental impacts of global development (Dean & McMullen 2007) and more environmentally sensitive products and services: representing an opportunity for the development of ventures that seek to satisfy this demand through entrepreneurial action. These ventures may choose to ''make do'' with existing resources in developing resource combinations that produce the least impact on the environment. The continuous conflict between the greater requirements for resources and limited resource availability in high potential sustainable ventures, with the added complexity of balancing this with an uncompromising focus on using ''what's on hand'' to lessen environment impacts may make bricolage behaviours critical for these ventures. Research into bricolage behaviour is however, the exception rather than the rule (Cunha 2005). More research is therefore needed to further develop and extend this emerging concept, especially in the context of sustainability ventures who are committed to personal and social goals of resourcefulness. To date, however, bricolage has not been studied specifically among high potential sustainable ventures. This research seeks to develop an in depth understanding of the impact of internal and external constraints and their combinations on the mechanisms employed in bricolage behaviours in differing dynamic environments. The following research question was developed to investigate this: How do internal, external resource constraints (or their combinations) impact bricolage resource decisions in high potential sustainability ventures? ---------- Methodology/Key Propositions: 6 case studies will be developed utilizing survey data from the Comprehensive Australian Study of Entrepreneurial Emergence (CAUSEE) large-scale longitudinal study of new venture start-ups in Australia. Prior to commencing case studies, 6 scoping interviews were conducted with key stakeholders including industry members, established businesses and government to ensure practical relevance in case development. The venture is considered the unit of analysis with the key informant being the entrepreneur and other management team members where appropriate. Triangulation techniques are used in this research including semi-structured interviews, survey data, onsite visits and secondary documentation website analysis, resumes, and business plans. These 6 sustainability ventures have been selected based on different environmental dynamism conditions including a traditionally mature market (building industry) and a more dynamic, evolving industry (renewable energy/solar ventures). In evaluating multidisciplinary literature, we expect the following external constraints are critical including: technology constraints (seen through lock-in of incumbents existing technology), institutional regulation and standards, access to markets, knowledge and training to nascent and young venture bricolage processes. The case studies will investigate internal constraints including resource fungability, resource combination capabilities, translating complex science/engineering knowledge into salient, valuable market propositions, i.e. appropriate market outcomes, and leveraging relationships may further influence bricolage decisions. ---------- Results and Implications: Intended ventures have been identified within the CAUSEE sample and have agreed to participate and secondary data collection for triangulation purposes has already commenced. Data collection of the case studies commenced 27th of May 2009. Analysis is expected to be completed finalised by 25th September 2009. This paper will report on the pattern of resource constraints and its impact on bricolage behaviours: its subsequent impact on resource deployment within venture creation and venture growth. As such, this research extends the theory of bricolage through the systematic analysis of constraints on resource management processes in sustainability ventures. For practice, this research may assist in providing a better understanding of the resource requirements and processes needed for continued venture persistence and growth in sustainability ventures. In these times of economic uncertainty, a better understanding of the influence on constraints and bricolage: the interplay of behaviours, processes and outcomes may enable greater venture continuance and success.

Trailblazers

A recent study in the United Kingdom (Ofsted Report 2008) provides strong evidence that well-organized activities outside the classroom contribute significantly to the quality and depth of children's learning, including their personal, social, and emotional development. Outdoor math trails supply further evidence of such enhanced learning: They are meaningful, stimulating, challenging, and exciting for children. Most important, these trails invite all students, irrespective of their classroom achievement level, to participate successfully in the problem activities and gain a sense of pride in the mathematics they create. Additionally, Math trails empower lifelong learning. Integrating "outside" mathematics with "inside" classroom mathematics can sow the seeds to develop flexible, creative, future-oriented mathematical thinkers and problem solvers. Here, English et al discuss how to design and implement math trails to promote active, meaningful, real-world mathematical learning beyond the classroom walls.

Placing the creative sector in innovation : the full gamut

This paper examines the place of the creative sector -- the arts, design, media and communications -- within the framework of contemporary innovation. The historical focus on science-and-technology by innovation policy makers has spurred many within the creative sector to argue how and why it also contributes to innovation. Drawing on a wide range of English-speaking research and policy documents, the full gamut of places for the creative sector in innovation is surveyed. The paper ends by scoping out the conceptual and empirical research that is required if ideas about innovation in the creative sector are to take up a mature position within innovation studies and related policy.

Managing the winds of change

Environmental impacts caused during Australia's comparatively recent settlement by Europeans are evident. Governments (both Commonwealth and States) have been largely responsible for requiring landholders – through leasehold development conditions and taxation concessions – to conduct clearing that is now perceived as damage. Most governments are now demanding resource protection. There is a measure of bewilderment (if not resentment) among landholders because of this change. The more populous States, where most overall damage has been done (i.e. Victoria and New South Wales), provide most support for attempts to stop development in other regions where there has been less damage. Queensland, i.e. the north-eastern quarter of the continent, has been relatively slow to develop. It also holds the largest and most diverse natural environments. Tree clearing is an unavoidable element of land development, whether to access and enhance native grasses for livestock or to allow for urban developments (with exotic tree plantings). The consequences in terms of regulations are particularly complex because of the dynamic nature of vegetation. The regulatory terms used in current legislation – such as 'Endangered' and 'Of concern' – depend on legally-defined, static baselines. Regrowth and fire damage are two obvious causes of change. A less obvious aspect is succession, where ecosystems change naturally over long timeframes. In the recent past, the Queensland Government encouraged extensive tree-clearing e.g. through the State Brigalow Development Scheme (mostly 1962 to 1975) which resulted in the removal of some 97% of the wide-ranging mature forests of Acacia harpophylla. At the same time, this government controls National Parks and other reservations (occupying some 4% of the State's 1.7 million km2 area) and also holds major World Heritage Areas (such as the Great Barrier Reef and the Wet Tropics Rainforest) promulgated under Commonwealth legislation. This is a highly prescriptive approach, where the community is directed on the one hand to develop (largely through lease conditions) and on the other to avoid development (largely by unusable reserves). Another approach to development and conservation is still possible in Queensland. For this to occur, however, a more workable and equitable solution than has been employed to date is needed, especially for the remote lands of this State. This must involve resident landholders, who have the capacity (through local knowledge, infrastructure and daily presence) to undertake most costeffectively sustainable land-use management (with suitable attention to ecosystems requiring special conservation effort), that is, provided they have the necessary direction, encouragement and incentive to do so.

Water sensitive urban design in landscape projects in Shijiazhuang and Yueyang in China [水敏性城市设计 (WSUD) 策略及景观项目中的应用 (中国石家莊和中國岳陽)]

With the rapid urbanization progress, water resources protection and water pollution control have become key problems of human environment construction and social sustainable development. Many countries, especially Australia, have mature experiences. Water Sensitive Urban Design (WSUD) is one of the successful strategies that is put forward under this global situation and helps releasing heavy environmental pressure from urbanization. The paper discussed main principles of WSUD and then took Shijiazhuang, Heibei and Yueng, Hunan for examples trying to apply WSUD in river landscape projects in China's new urban area, thus doing contributions to more sustainable water management in new urban areas in China.

Pre-service teacher education and the development of middle school teacher identity : an exploratory study

This research study investigated the factors that influenced the development of teacher identity in a small cohort of mature-aged graduate pre-service teachers over the course of a one-year Graduate Diploma program (Middle Years). It sought to illuminate the social and relational dynamics of these pre-service teachers’ experiences as they began new ways of being and learning during a newly introduced one-year Graduate Diploma program. A relational-ontological perspective underpinned the relational-cultural framework that was applied in a workshop program as an integral part of this research. A relational-ontological perspective suggests that the development of teacher identity is to be construed more as an ontological process than an epistemological one. Its focus is more on questions surrounding the person and their ‘becoming’ a teacher than about the knowledge they have or will come to have. Hence, drawing on work by researchers such as Alsup (2006), Gilligan, (1982), Isaacs, (2007), Miller (1976), Noddings, (2005), Stout (2001), and Taylor, (1989), teacher identity was defined as an individual pre-service teacher’s unique sense of self as a teacher that included his or her beliefs about teaching and learning (Alsup, 2006; Stout, 2001; Walkington, 2005). Case-study was the preferred methodology within which this research project was framed, and narrative research was used as a method to document the way teacher identity was shaped and negotiated in discursive environments such as teacher education programs, prior experiences, classroom settings and the practicum. The data that was collected included student narratives, student email written reflections, and focus group dialogue. The narrative approach applied in this research context provided the depth of data needed to understand the nature of the mature-aged pre-service teachers’ emerging teacher identities and experiences in the graduate diploma program. Findings indicated that most of the mature-aged graduate pre-service teachers came in to the one-year graduate diploma program with a strong sense of personal and professional selves and well-established reasons why they had chosen to teach Middle Years. Their choice of program involved an expectation of support and welcome to a middle-school community and culture. Two critical issues that emerged from the pre-service teachers’ narratives were the importance they placed on the human support including the affirmation of themselves and their emerging teacher identities. Evidence from this study suggests that the lack of recognition of preservice teachers’ personal and professional selves during the graduate diploma program inhibited the development of a positive middle-school teacher identity. However, a workshop program developed for the participants in this research and addressing a range of practical concerns to beginning teachers offered them a space where they felt both a sense of belonging to a community and where their thoughts and beliefs were recognized and valued. Thus, the workshops provided participants with the positive social and relational dynamics necessary to support them in their developing teacher identities. The overall findings of this research study strongly indicate a need for a relational support structure based on a relational-ontological perspective to be built into the overall course structure of Graduate Pre-service Diplomas in Education to support the development of teacher identity. Such a support structure acknowledges that the pre-service teacher’s learning and formation is socially embedded, relational, and a continual, lifelong process.

Heavy vehicle suspensions : testing and analysis

Transport regulators consider that, with respect to pavement damage, heavy vehicles (HVs) are the riskiest vehicles on the road network. That HV suspension design contributes to road and bridge damage has been recognised for some decades. This thesis deals with some aspects of HV suspension characteristics, particularly (but not exclusively) air suspensions. This is in the areas of developing low-cost in-service heavy vehicle (HV) suspension testing, the effects of larger-than-industry-standard longitudinal air lines and the characteristics of on-board mass (OBM) systems for HVs. All these areas, whilst seemingly disparate, seek to inform the management of HVs, reduce of their impact on the network asset and/or provide a measurement mechanism for worn HV suspensions. A number of project management groups at the State and National level in Australia have been, and will be, presented with the results of the project that resulted in this thesis. This should serve to inform their activities applicable to this research. A number of HVs were tested for various characteristics. These tests were used to form a number of conclusions about HV suspension behaviours. Wheel forces from road test data were analysed. A “novel roughness” measure was developed and applied to the road test data to determine dynamic load sharing, amongst other research outcomes. Further, it was proposed that this approach could inform future development of pavement models incorporating roughness and peak wheel forces. Left/right variations in wheel forces and wheel force variations for different speeds were also presented. This led on to some conclusions regarding suspension and wheel force frequencies, their transmission to the pavement and repetitive wheel loads in the spatial domain. An improved method of determining dynamic load sharing was developed and presented. It used the correlation coefficient between two elements of a HV to determine dynamic load sharing. This was validated against a mature dynamic loadsharing metric, the dynamic load sharing coefficient (de Pont, 1997). This was the first time that the technique of measuring correlation between elements on a HV has been used for a test case vs. a control case for two different sized air lines. That dynamic load sharing was improved at the air springs was shown for the test case of the large longitudinal air lines. The statistically significant improvement in dynamic load sharing at the air springs from larger longitudinal air lines varied from approximately 30 percent to 80 percent. Dynamic load sharing at the wheels was improved only for low air line flow events for the test case of larger longitudinal air lines. Statistically significant improvements to some suspension metrics across the range of test speeds and “novel roughness” values were evident from the use of larger longitudinal air lines, but these were not uniform. Of note were improvements to suspension metrics involving peak dynamic forces ranging from below the error margin to approximately 24 percent. Abstract models of HV suspensions were developed from the results of some of the tests. Those models were used to propose further development of, and future directions of research into, further gains in HV dynamic load sharing. This was from alterations to currently available damping characteristics combined with implementation of large longitudinal air lines. In-service testing of HV suspensions was found to be possible within a documented range from below the error margin to an error of approximately 16 percent. These results were in comparison with either the manufacturer’s certified data or test results replicating the Australian standard for “road-friendly” HV suspensions, Vehicle Standards Bulletin 11. OBM accuracy testing and development of tamper evidence from OBM data were detailed for over 2000 individual data points across twelve test and control OBM systems from eight suppliers installed on eleven HVs. The results indicated that 95 percent of contemporary OBM systems available in Australia are accurate to +/- 500 kg. The total variation in OBM linearity, after three outliers in the data were removed, was 0.5 percent. A tamper indicator and other OBM metrics that could be used by jurisdictions to determine tamper events were developed and documented. That OBM systems could be used as one vector for in-service testing of HV suspensions was one of a number of synergies between the seemingly disparate streams of this project.

The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.